In general, stem cells are undifferentiated cells which can give rise to a succession of mature functional cells. For example, a haematopoietic stem cell may give rise to any of the different types of terminally differentiated blood cells. Embryonic stem (ES) cells are derived from the embryo and are pluripotent, thus possessing the capability of developing into any organ, cell type or tissue type or, at least potentially, into a complete embryo. ES cells may be derived from the inner cell mass of the blastocyst, which have the ability to differentiate into tissues representative of the three embryonic germ layers (mesoderm, ectoderm, endoderm), and into the extra-embryonic tissues that support development.
Human embryonic stem cells (hES cells) are pluripotent cell lines derived from the inner cell mass of the blastocyst. These cells have the ability to differentiate into functional tissues representative of the three embryonic germ layers (mesoderm, ectoderm, endoderm), and into extra-embryonic tissues that support development. Because of their ability to generate these different cellular fates, hES cells are considered to be of great potential for future therapies.
However, during routine culture in vitro, established hES cell lines have a tendency to spontaneously differentiate. Because the pluripotency of these cells is associated with their undifferentiated state, it is desirable to find a way to limit this spontaneous differentiation. Contrary to what is seen in mouse embryonic stem cells, leukemia inhibitory factor (LIF) does not prevent the spontaneous differentiation of hES cells [1]. Thus, a common way to grow and then to maintain hES cells in an optimum state is to cultivate them on feeder layers, which are constituted by primary mouse embryonic fibroblasts (MEF), in media supplemented with high doses of fetal calf serum.
However, serum contains a wide variety of biologically active compounds that might have the potential to adversely affect hES cell growth and differentiation. Furthermore, there is a biosafety issue if cells cultured in animal serum are subsequently used for implantation in a human or for the production of a biological therapeutic.
With regard to these issues and in order to establish a serum-free culture system to grow hES cells, it is of great importance to identify the specific factors in serum that are responsible for its beneficial effect on the growth of hES cells. Thus, alternative approaches to traditional culture systems are desirable, such as the use of a serum replacement medium such as Knockout Serum Replacement [2, 3].
Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are two small bioactive lysophospholipids, present in serum (at concentration of up to 1 and 5 μM respectively) [4], released by activated platelets, which act on a wide range of cell types derived from the three developmental germ layers. Most of the effects of these lysophospholipids seem to be mediated by specific lysophospholipid G-protein coupled receptors (LPL receptors) previously named endothelial differentiation gene (Edg) receptors.
Up to now, eight distinct mammalian LPL/Edg receptors have been identified: S1P1/Edg-1, S1P2/Edg-5, S1P3/Edg-3, S1P4/Edg-6 and S1P5/Edg-8 are specific for S1P while LPA1/Edg-2, LPA2/Edg-4 and LPA3/Edg-7 are specific for LPA (for reviews see [5, 6]). Each of these receptors is coupled to at least one G protein and can activate or inhibit specific signaling pathways. For instance, all these receptors are coupled to Gi/o proteins (for review see [5, 6]).
By activating notably these Gi/o proteins, S1P and LPA can stimulate the extracellular-signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein (MAP) kinase family, and thus are involved in regulation of major cellular events, such as cell proliferation or differentiation. S1P and LPA are potent biological agents involved in numerous cell events, such as proliferation, differentiation, death or migration (for review see [5]) since the very early stages of development.
S1P stimulates mammalian angiogenesis, at least via S1P, and S1P2 [7-10]. Thus, S1P1, knockout mice show impaired blood vessel maturation. Moreover, in the zebrafish, S1P is required for normal heart development [11]. Thus, in these animals, the mutation of the gene mil that encodes the S1P receptor Mil (very similar to the mammalian S1P2 receptor) impairs migration of cardiac progenitor cells [11].
On the other hand, LPA seems to be mainly involved in neurogenesis [12]. For instance, LPA, probably via LPA1, stimulates cell cycle-morphological changes and cell migration of cultured cortical neuroblasts. Moreover, LPA, probably via LPA2, regulates the migration of post-mitotic neurons to their final destination. Last but not least, LPA, knockout mice present abnormal cerebral cortices and olfactory bulbs, probably due to impaired development, demonstrating LPA, is essential for a normal brain development [13].
Within serum, Platelet-Derived Growth Factor (PDGF) is a major protein growth factor that has been widely described as a potent mitogen of numerous kinds of cells. PDGF has also been shown to induce chemotaxis, actin re-organization, and to prevent apoptosis. This growth factor belongs to a family of dimeric isoforms of polypeptide chains, A, B, C and D that act through different tyrosine kinase receptors: PDGFR-α and PDGFR-β.
S1P and PDGF have additional effects that induce biological responses. Thus S1P and PDGF are able to regulate smooth muscle cell migration, proliferation and vascular maturation. Moreover, Hobson et al. (2001), and Rosenfeld et al. (2001) demonstrated that PDGF-stimulated cell motility is S1P1-dependent in HEK 293 cells and MEF [14, 15] while Kluk et al. (2003) showed that this effect was independent of S1P, in vascular smooth muscles and MEF [16]. Last but not least, it is now proposed that PDGF is able to stimulate the enzyme sphingosine kinase, which leads to an increase in S1P intracellular concentration [17], an effect that would be vascular smooth muscle cells [18].
The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.